Adrenal ascorbate-dependent cytochrome (cyt.) b561 is a transmembrane electron transporter required for synthesis of catecholamine and some peptide hormone and is the prototype of an expanding and insufficiently characterized cyt. b561 protein family; other mammalian family members have been linked to intestinal iron transport and tumor suppression. Our overall goal is to characterize the mechanism of adrenal b561 and to use information about this cytochrome to understand the structure and function of other b561 family members. The specific aims of this project are: 1) Define the structural kernel of adrenal cyt. b561, determine its relationship to the membrane bilayer, and evaluate the kernel's generality among mammalian cyt. b561 family members; 2) Characterize the interaction of adrenal cyt. b561 with its natural reductant, ascorbate, and its natural oxidant, semidehydroascorbate; and 3) Determine the path of electron flow via adrenal cyt. b561 and its regulation. Planned experiments will be conducted on the native mammalian protein and recombinant wild type and mutant forms of the adrenal cytochrome and other mammalian cyt. b561 family members in prokaryotic and eukaryotic systems that we have developed. We will characterize the topological, structural, and kinetic properties of these recombinant proteins and purified endogenous adrenal cyt b561 using biochemical and biophysical techniques. Understanding how adrenal cyt. b561 functions is a fundamental part of understanding the physiology and pathology of epinephrine and norepinephrine and of some peptide hormones in humans. Defining similarities and differences between adrenal cyt. b561 and recently-discovered human cyt. b561 family members will accelerate characterization of the physiological and pathological roles of the newer cyt. b561s. [unreadable] [unreadable] [unreadable]